The long term goal of the proposed research is to elucidate critical steps in the molecular mechanism of homologous genetic recombination. This goal will be approached by studying hotspots of recombination, which stimulate a critical rate limiting step of recombination. The Chi hotspot of the bacterium Escherichia coli and the M26 hotspot of the fission yeast Schizosaccharomyces pombe will be investigated. The proteins that interact with these sites will also be investigated: the RecBCD enzyme with Chi, and the Atfl.Pcrl transcription factor with M26. With both hotspots a key question concerns the DNA reaction at the hotspot, especially in intact cells undergoing recombination. With M26 an additional important question concerns the reciprocal interactions of M26 with its surrounding chromosomal context. Answers to these questions will reveal the rate-limiting steps of recombination and the regulation of recombination both spatially along chromosomes and temporally during the organism's life cycle. The specific aims are 1) to determine the change of RecBCD enzyme at Chi, the reaction at Chi in intact cells, and the factors that limit Chi-stimulated recombination, and 2) to elucidate how Atfl.Pcrl activates M26 and how the hotspot activity of M26 depends on its chromosomal context and on meiosis. These aims will be achieved by a combination of biochemistry with purified components, and genetics with intact cells. Recombination is important in the faithful repair of double-strand DNA breaks in chromosomes and in the faithful segregation of chromosomes during meiosis. Aberrancies of recombination may be responsible for chromosomal rearrangements associated with cancer, birth defects, and certain hereditary diseases. Gene therapy by homologous gene replacement and gene targeting will be facilitated by understanding the molecular mechanism of homologous recombination and its stimulation by hotspots.